Project Description. GABAA receptor mediated inhibition sculpts neuronal activity, plays key roles in mood and cognition, and is a target of important drugs including ethanol. Major classes of GABAA receptors are defined by ? vs. ? subunit presence, but the relative contributions of these two receptor classes to inhibition and to ethanol effects remain unclear. Classically, ? populations mediate phasic inhibition, while ? populations mediate tonic inhibition. A recurring hypothesis is that ethanol at low concentrations selectively potentiates GABA actions at ? receptors. However, this idea has remained controversial and unresolved. Environmental factors including other subunits and post-translational modifications complicate efforts to test the hypothesis rigorously. A key barrier has been the inability to isolate the two receptor populations in native cells without complications arising from genetic manipulations associated with previous studies (e.g., compensation for genetic deletion). Pharmacological isolation of ? and ? receptors would allow direct study of ethanol selectivity with unprecedented resolution and rigor. We approached this issue through a knock- in/chemogenetic strategy. We developed two knock-in mouse lines harboring picrotoxin resistance in the endogenous ? or ? subunit respectively. Proof-of-principle experiments in hippocampal dentate granule neurons have demonstrated the ability to isolate the respective receptor populations and have led to the hypothesis that ? may play a larger than expected role in phasic inhibition. We will first test the generality of this conclusion by repeating the evaluation in two additional cell types proposed to be critical for acute ethanol effects: medium spiny neurons of the nucleus accumbens and cerebellar granule neurons. We then proceed to test the hypothesis that ? receptors are selectively sensitive to low ethanol concentrations, compared with ? receptors and with receptors devoid of either ? or ?. Conventional inducible knockouts are used to validate results. Our experiments will help explain the acute intoxicating effects of ethanol, and success of our exploratory studies would also prompt future studies to explore chronic effects of ethanol, for which GABAA receptor populations have also been implicated.